LPS vaccine

ABSTRACT

A vaccine composition for birds comprising as an active ingredient a structure containing O-antigen derived from Gram-negative bacteria, provided that said structure does not contain a whole cell, and a process for preparing the same are provided. By using a structure containing O-antigen (e.g. lipopolysaccharide) derived from Gram-negative bacteria as an active ingredient in accordance with the present invention, alleviation of inoculation reaction and reduction in an amount of injection are attained as compared to the conventional whole-cells vaccine to thereby allow for the increase in the number of other antigens to be mixed therewith.

This application is a continuation of U.S. application Ser. No.15/098,563 filed Apr. 14, 2016, which is and is a divisional of U.S.application Ser. No. 14/385,606 filed Sep. 16, 2014, now U.S. Pat. No.9,345,756, and incorporated herein by reference, which is a NationalStage application of PCT/JP2013/054038 filed Feb. 19, 2013 and claimsthe benefit of JP 2012-065162 filed Mar. 22, 2012.

TECHNICAL FIELD

The present invention provides a novel vaccine useful for preventinginfectious diseases caused by Gram-negative bacteria such as Salmonellaand E. coli. Specifically, the present invention relates to a method ofimmunizing non-human animals, in particular, birds using as a protectiveantigen a structure containing O-antigen (e.g. lipopolysaccharide)derived from Gram-negative bacteria.

BACKGROUND ART

Bacteria have different dye affinity depending upon differentcompositions of the cell wall when tested with Gram staining and aredivided into two major groups of Gram-negative and Gram-positivebacteria based on differences in their dye affinity. In case ofGram-negative bacteria, the cell wall consists of an outer membrane anda peptidoglycan layer on the inside of the outer membrane. The outermembrane consists of phospholipids, lipopolysaccharides (LPS),lipoproteins and membrane proteins. A unit membrane structure of theouter membrane consists of phospholipids and lipopolysaccharides. Alipopolysaccharide is found in an outer layer of the membrane and aphospholipid in an inner layer thereof. A lipopolysaccharide consists ofa high molecular weight lipid, called Lipid A, and a polysaccharidebound thereto. Lipid A makes up an outer layer of the outer membranewhereas a polysaccharide extends outwards from the outer membrane. Thekind of saccharides is different from each other between the outerportion and the portion at the vicinity of Lipid A. The outer portion iscalled an O-side chain polysaccharide (O-antigen) and the inner portionis called a core polysaccharide. The saccharide consisting of O-antigenis chiefly a hexose and a pentose and a basic structure consisting of 3to 5 kinds of these saccharides appear repeatedly. In the corepolysaccharide are present, in addition to these saccharides,saccharides unique to the respective bacteria such as an octose, e.g.ketodeoxyoctonate, and a heptose. Lipid A is a lipid unique to therespective bacteria which comprises a saccharide, i.e. two molecules ofglucosamine which are β-1,6-bound to each other, and phosphoric acid anda fatty acid bound to said saccharide. Lipid A is bound to a corepolysaccharide at 6′-position of the saccharide.

There are many kinds of Gram-negative bacteria. Salmonella and E. coli,belonging to Enterobacteriaceae, and Haemophilus, belonging toPasteurella, are also included in Gram-negative bacteria.

Salmonella, secondary large bacillus with peritrichous flagella, isdivided into groups by the type of O-antigen and is further subdividedby the types of H-antigen, resulting in more than 2,000 kinds ofserotype. A host range of Salmonella is quite wide and a variety ofmammals including human and birds are known to be infected with or tohold Salmonella. When chicken are infected, Salmonella may cause septicdiseases in young chicks. In case of adult chickens, however, carrierchickens are asymptomatic to escape culling and as a result chicken meatand eggs derived from chickens contaminated with Salmonella aredistributed to induce food poisoning in human through food productsmanufactured thereby.

Food poisoning by Salmonella develops after a latent period of 12 to 48hours after intake of contaminated food. A latent period may varydepending on an intake amount of bacteria, the condition and age ofpatients. Symptoms are mainly acute gastroenteritis and cardinalsymptoms are diarrhea, abdominal pain, vomiting and fever. Thus, asalmonella vaccine for chicken is not preventing chickens from thedisease onset but is an important vaccine used for public health.

Among conventional vaccines against Salmonella is a whole-cells vaccinewhich comprises cells of inactivated Salmonella. A whole-cells vaccine,however, may cause side effects since it contains portions that are notof antigenicity. In case of chickens that are bred in flocks, in view oflabor saving of vaccination, there is a strong demand for a multivalentvaccine that may prevent many diseases with a single injection. Also, amultivalent vaccine may contribute to reduction of stress in chickenssince it reduces the number of injections. However, although amultivalent vaccine has such convenience, it is liable to causevaccination reaction at the injection site, in particular, when itcontains bacteria such as Salmonella.

Under the circumstances, research of a component vaccine againstSalmonella has been conducted, among which application of alipopolysaccharide (O-antigen) is studied. For instance, there is areport that a conjugate consisting of O-antigen derived from SalmonellaTyphimurium bound to a carrier protein is administered to mice toconfirm its efficacy (Non-patent reference 1). There is also a reportthat a lipopolysaccharide derived from Salmonella Typhi is administeredto mice to confirm its efficacy (Patent reference 1). Other thanSalmonella, there is a report that a lipopolysaccharide derived fromBurkholderia thailandensis or Burkholderia pseudomallei is administeredto mice to confirm its efficacy (Patent reference 2, Non-patentreference 2).

However, contamination of a lipopolysaccharide is avoided withmeticulous care in a medicament used for living body since alipopolysaccharide may clinically cause a variety of highly lethaldiseases such as septic shock, disseminated intravascular coagulation(DIC) and multiple organ failure (MOF) and may become a causal factor offever even in a trace amount (Non-patent reference 3). In practice, itis reported that more than 90% of mice die within 72 hours whenadministered with a lipopolysaccharide (Patent reference 3). Under thecircumstances, an idea to use a lipopolysaccharide per se as a vaccineantigen would not arise generally.

PATENT REFERENCES

-   Patent reference 1: WO2004/052394-   Patent reference 2: WO2010/082020-   Patent reference 3: Japanese Patent Publication No. 2001-26602

NON-PATENT REFERENCES

-   Non-patent reference 1: Infect. Immun, 60, pp 4679-4686, 1992-   Non-patent reference 2: Vaccine, 28, pp 7551-7555, 2010-   Non-patent reference 3: Bull. Natl. Inst. Health Sci., 126, pp    19-33, 2008-   Non-patent reference 4: AVIAN DISEASES, 53, pp 281-286, 2009

DISCLOSURE OF THE INVENTION

(Technical Problem to be Solved by the Invention)

The present invention aims at making it possible to produce amultivalent vaccine and a mixed vaccine for a vaccine againstGram-negative bacteria such as Salmonella and E. coli and at reducinginoculation reaction.

(Means for Solving the Problems)

The present inventors have earnestly studied the problems mentionedabove and as a result have found that swelling at the injection site isunexpectedly restrained when a vaccine comprising as an activeingredient a structure containing O-antigen (e.g. lipopolysaccharide)derived from Gram-negative bacteria is administered to birds. Namely, astructure containing O-antigen is released from cells contained inculture of Gram-negative bacteria (e.g. Salmonella), e.g. by sonicationor phenol treatment, and then is collected with a column to which saidstructure is adhered to. A vaccine composition comprising as an activeingredient the aforementioned structure is then prepared via a step ofemulsification, if necessary. The present inventors have found that thethus prepared vaccine composition may confer immunization against saidGram-negative bacteria to birds (e.g. chicken) when immunized therewithwith no specific side effects to thereby complete the present invention.

The present invention includes the following inventions.

[1] A vaccine composition for birds comprising as an active ingredient astructure containing O-antigen derived from Gram-negative bacteria,provided that said structure does not contain a whole cell.

[2] The vaccine composition according to [1] wherein the structurecontaining O-antigen is lipopolysaccharide.

[3] The vaccine composition according to [1] or [2] whereinGram-negative bacteria are Enterobacteriaceae or Pasteurella.

[4] The vaccine composition according to [3] wherein Enterobacteriaceaeis Salmonella or E. coli.

[5] The vaccine composition according to [4] wherein Salmonella is oneor more selected from the group consisting of O4, O7 and O9 Groups.

[6] The vaccine composition according to [4] or [5] wherein Salmonellais one or more selected from the group consisting of SalmonellaEnteritidis, Salmonella Typhimurium and Salmonella Infantis.

[7] The vaccine composition, according to [4] or [5] wherein thestructure containing O-antigen derived from Gram-negative bacteria is amixture of structures containing O-antigen derived from SalmonellaEnteritidis, Salmonella Typhimurium and Salmonella Infantis.[8] The vaccine composition according to [4] wherein E. coli is E. colihaving O-antigen of O78.

The vaccine composition according to [3] wherein Pasteurella isHaemophilus.

[10] The vaccine composition according to [9] wherein Haemophilus isHaemophilus paragallinarum.

[11] The vaccine composition according to any one of [1] to [10] whereinthe structure containing O-antigen is a structure containing O-antigencontained at at least 5,400 EU/ml from each Salmonella in the vaccinecomposition.

[12] The vaccine composition according to any one of [1] to [11] whereinthe vaccine composition further comprises an antigen derived from one ormore pathogens selected from the group consisting of Newcastle diseasevirus, avian infectious bronchitis virus, Mycoplasma gallisepticum, Eggdrop syndrome virus and Haemophilus paragallinarum.[13] A process for preparing a vaccine composition for birds comprisingas an active ingredient a structure containing O-antigen derived fromGram-negative bacteria, the process comprising the steps (1) to (4) asfollows:(1) a step of culturing Gram-negative bacteria (step of culture);(2) a step of releasing the aforementioned structure from cellscontained in the solution after said culture (step of release);(3) a step of collecting the aforementioned structure from the solutionafter said release (step of collection); and(4) a step of preparing the solution after said collection to obtain avaccine composition (step of preparation).[14] The process according to [13] wherein the step of releasing theaforementioned structure is a step of sonication or a step of treatmentwith phenol.[15] The process according to [13] or [14] wherein the process furthercomprises a step of inactivating cells (step of inactivation) and/or astep of emulsifying the aforementioned structure (step ofemulsification).[16] The process according to [15] wherein the step of inactivation iscarried out after the step of culture and the step of emulsification iscarried out after the step of collection.[17] The process according to any one of [13] to [16] wherein theprocess further comprises a step of measuring an amount of theaforementioned structure (step of measurement).[18] The process according to [17] wherein the step of measurement iscarried out after the step of collection.[19] The process according to any one of [13] to [18] wherein theprocess further comprises a step of removing Lipid A (step of removingLipid A).[20] The process according to [19] wherein the step of removing Lipid Ais carried out after the step of collection.

EFFECTS OF THE INVENTION

By using a structure containing O-antigen (e.g. lipopolysaccharide)derived from Gram-negative bacteria as an active ingredient of a vaccinecomposition, it becomes possible to alleviate swelling at the injectionsite as compared to the conventional whole-cells vaccine. Besides, bypreparing a component vaccine, it becomes possible to further increasethe number of other antigens to be mixed therewith without increasing anamount of injection.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows a swelling score at the injection site for a mixed vaccineof lipopolysaccharides derived from Salmonella Enteritidis, SalmonellaInfantis and Salmonella Typhimurium (trivalent LPS vaccine) or a vaccinecomprising other antigens in addition to the trivalent LPS vaccine(10-valent mixed LPS vaccine).

FIG. 2 shows the number of cells shed when a challenge test withSalmonella Typhimurium is carried out for trivalent LPS vaccine whereinthe asterisk * shows a significant difference from control group(p<0.05).

FIG. 3 shows the number of cells shed when a challenge test withSalmonella Enteritidis is carried out for trivalent LPS vaccine whereinthe asterisk * shows a significant difference from control group(p_(<)0.05).

FIG. 4 shows the number of cells shed when a challenge test withSalmonella Infantis is carried out for trivalent LPS vaccine wherein theasterisk * shows a significant difference from control group (p<0.05).

FIG. 5 shows the number of cells shed when a challenge test withSalmonella Enteritidis is carried out for 10-valent mixed LPS vaccinewherein the asterisk * shows a significant difference from control group(p<0.05).

FIG. 6 shows the number of cells shed when a challenge test withSalmonella Infantis is carried out for 10-valent mixed LPS vaccinewherein the asterisk * shows a significant difference from control group(p<0.05).

BEST MODE FOR CARRYING OUT THE INVENTION

1. Vaccine Composition

The first embodiment of the present invention is a vaccine compositionfor birds comprising as an active ingredient a structure containingO-antigen derived from Gram-negative bacteria.

In accordance with the present invention, in a vaccine compositioncomprising as an active ingredient a structure containing O-antigen(specifically, lipopolysaccharide) derived from Salmonella Typhimurium,Salmonella Infantis or Salmonella Enteritidis, the shedding of cells isreduced against challenge with the respective cells. It is assumed to bedue to maintenance of antigenicity against the respective structuresderived from a plurality of Gram-negative bacteria even if therespective structures are mixed together. Besides, in the 10-valentmixed vaccine comprising the trivalent vaccine mixed with antigensdifferent from the aforementioned structures, antigenicity against cellsthat provided the aforementioned structures is maintained. It is assumedto be due to maintenance of antigenicity against the aforementionedstructures even if the mixed vaccine comprises antigens other than theaforementioned structures.

As such, the vaccine composition of the present invention includes:

(A) a vaccine composition comprising as an active ingredient theaforementioned structure derived from a single Gram-negative bacteria(hereinafter also referred to as “monovalent vaccine”);

(B) a vaccine composition comprising as an active ingredient theaforementioned structure derived from a plurality of Gram-negativebacteria (hereinafter also referred to as “polyvalent vaccine”); and

(C) a vaccine composition comprising a monovalent vaccine or apolyvalent vaccine together with antigens different from theaforementioned structure (hereinafter also referred to as “mixedvaccine”).

(1) Bacterial Cells

The vaccine composition of the present invention comprises as an activeingredient a structure containing O-antigen. Thus, the vaccinecomposition of the present invention may widely be applied toGram-negative bacteria having the aforementioned structure. Forinstance, Gram-negative bacteria as used in the present invention mayinclude Gram-negative facultatively anaerobic rod, Anaplasmataceae,Arcobacter, Bartonellaceae, Brachyspira, Buchnera, Campylobacter,Chlamydiales, Chloroflexus, Gram-negative aerobic bacteria,Gram-negative anaerobic bacteria, Gram-negative oxygenic photosyntheticbacteria, Helicobacter, Lawsonia bacteria, Methylosinus,Oceanospirillaceae, Ornithobacterium, Piscirickettsiaceae, Rhodobacter,Rhodomicrobium, Rhodovulum, Rickettsiaceae, Roseobacter, Spirillaceae,and Tenericutes, preferably Gram-negative facultatively anaerobic rod.

Gram-negative facultatively anaerobic rod may includeEnterobacteriaceae, Pasteurellaceae, Actinobacillus, Aeromonadaceae,Azoarcus, Capnocytophaga, Cardiobacteriaceae, Chromobacterium,Eikenella, Gardnerella, Moritella, Rahnella, Shewanella,Streptobacillus, Vibrionaceae, and Zymomonas, preferablyEnterobacteriaceae and Pasteurellaceae.

Enterobacteriaceae may include Salmonella, Escherichia,Calymmatobacterium, Citrobacter, Edwardsiella, Enterobacter, Erwinia,Hafnia, Klebsiella, Kluyvera, Morganella, Pantoea, Pectobacterium,Photorhabdus, Plesiomonas, Proteus, Providencia, Serratia, Shigella,Wigglesworthia, Xenorhabdus, and Yersinia, preferably Salmonella andEscherichia.

Salmonella is divided into groups by the types of O-antigen and isfurther subdivided by the types of H-antigen, including O2 Group (A), O4Group (B), O7 Group (C1, C4), O8 Group (C2, C3), O9 Group (D1), andO3/O10 Group (E1, E2, E3) as shown in Table 1 and Table 2 (the olddesignation shown in the parenthesis).

TABLE 1 O-Antigen: O-Serotype Serotype (optionally deleted) O2 (A)Salmonella Paratyphi A 1, 2, 12 O4 (B) Salmonella Paratyphi B 1, 4, (5),12 Salmonella Stanley 1, 4, (5), 12, 27 Salmonella Derby 1, 4, (5), 12Salmonella Agona 1, 4, 12 Salmonella Typhimurium 1, 4, (5), 12Salmonella Heidelberg 1, 4, (5), 12 O7 (C1, C4) Salmonella Paratyphi C6, 7, (Vi) Salmonella Choleraesuis 6, 7, Salmonella Braenderup 6, 7, 14Salmonella Montevideo 6, 7, 14 Salmonella Thompson 6, 7, 14 SalmonellaVirchow 6, 7 Salmonella Infantis 6, 7, 14 Salmonella Bareilly 6, 7, 14Salmonella Tennessee 6, 7, 14

TABLE 2 O-Antigen: O-Serotype Serotype (optionally deleted) O8 (C2, C3)Salmonella Narashino 6, 8 Salmonella Newport 6, 8, 20 SalmonellaLichfield 6, 8 O9 (D1) Salmonella Sendai 1, 9, 12 Salmonella Typhi 9,12, (Vi) Salmonella Enteritidis 1, 9, 12 Salmonella Panama 1, 9, 12Salmonella enterica 1, 9, 12 subsp. salamae Salmonella Gallinarum 1, 9,12 O3/O10 (E1, Salmonella Anatum 3, 10, (15), (15, 34) E2, E3)Salmonella Meleagridis 3, 10, (15), (15, 34) Salmonella London 3, 10,(15) Salmonella Give 3, 10, (15), (15, 34) Salmonella Weltevreden 3, 10,(15)

In accordance with the present invention, in a trivalent vaccinecomprising as an active ingredient a structure containing O-antigensderived from Salmonella Typhimurium (O4 Group), Salmonella Infantis (O7Group) and Salmonella Enteritidis (O9 Group), the shedding of cells isreduced against challenge with the respective cells. It is assumed to bedue to maintenance of antigenicity of the aforementioned structuresprepared by the process for preparation of the present inventionirrespective of the types of O-antigen. Thus, it is assumed that thevaccine composition of the present invention may be applicable toSalmonella as whole but not limited to Salmonella belonging to O4 Group,O7 Group and O9 Group.

In Non-Patent Reference 4, in birds immunized with a vaccine comprisingcells of Salmonella Typhimurium, Salmonella Infantis and SalmonellaEnteritidis, the shedding of cells is reduced against challenge not onlywith the respective vaccine strains but also with Salmonella Heidelbergbelonging to the same O-antigen group (O4 Group) as SalmonellaTyphimurium. Thus, it is assumed that the vaccine may also be effectivefor cells that have homologous O-antigen to that of the vaccine strainsbut are of different serotypes. Namely, in case of a Salmonella vaccine,it is assumed that the vaccine may also be effective for cells havinghomologous O-antigen to that of the vaccine strains, in other words, forcells of the same O-antigen Group as that of the vaccine strains.Therefore, the vaccine composition of the present invention, whenapplied for Salmonella, may also be applicable to the other Salmonellacells belonging to O4 Group, O7 Group and O9 Group.

Thus, the vaccine composition of the present invention includes:

(A) a monovalent vaccine comprising as an active ingredient a structurecontaining O-antigen derived from Salmonella;

(B) a polyvalent vaccine comprising as an active ingredient structurescontaining O-antigen derived from two or more Salmonella;

(C) a monovalent vaccine comprising as an active ingredient a structurecontaining O-antigen derived from Salmonella belonging to O4 Group, O7Group or O9 Group;

(D) a polyvalent vaccine comprising as an active ingredient structurescontaining O-antigen derived from two or more Salmonella selected fromthe group consisting of O4 Group, O7 Group and O9 Group; and

(E) a mixed vaccine comprising the above monovalent vaccine or thepolyvalent vaccine together with antigens different from theaforementioned structure.

Exemplary Salmonella included in Salmonella, O4 Group, 07 Group or O9Group may include the cells shown in Table 1 and Table 2. Preferably,Salmonella is those belonging to O4 Group, Group and O9 Group. Also,preferably, O4 Group is Salmonella Typhimurium, O7 Group is SalmonellaInfantis and O9 Group is Salmonella Enteritidis.

E. coli is classified by a combination of three antigens, O-antigen,K-antigen and H-antigen. About 160 types for O-antigen, about 100 typesfor K-antigen and about 56 types for H-antigen are known. Specific typesof E. coli may develop diarrhea and gastroenteritis and may be a causeof food poisoning. Such E. coli is generally classified intoenterotoxigenic E. coli, enteroinvasive E. coli, enterohaemorrhagic E.coli, enteropathogenic E. coli, enterotoaggregative E. coli anddiffusely adherent E. coli. Groups belonging to enterotoxigenic E. coliinclude O18, O26, O44, O55, O86, O111, O112, O114, O119, O125, O126,O127, O128a, and O142. Enterotoxigenic E. coli includes O4, O6, O7, O8,O9, O15, O18, O20, O25, O27, O63, O77, O78, O80, O85, O114, O115, O126,O128a, O139, O148, O153, O159, O167, O168, and O169. Enteroinvasive E.coli includes O28c, O29, O112a, O124, O136, O143, O144, O152, O164, andO167. Enterohaemorrhagic E. coli includes O1018, O26, O91, O111, O113,O114, O115, O117, O119, 0121, O128, O145, and O157. Such E. coli ispreferably O78.

Pasteurellaceae includes Haemophilus, Mannheimia and Pasteurella,preferably Haemophilus.

Haemophilus includes Haemophilus paragallinarum, Haemophilus ducreyi,Haemophilus influenzae, Haemophilus parainfluenzae, Haemophilusparaphrophilus, Haemophilus parasuis, and Haemophilus somnus, preferablyHaemophilus paragallinarum.

(2) Structure Containing O-Antigen

The vaccine composition of the present invention comprises as an activeingredient a structure containing O-antigen. Since O-antigen is mainlyresponsible for antigenicity, a structure containing O-antigen may beany structure as far as it contains O-antigen provided that it is astructure that does not contain a whole cell. For instance, a structurecontaining O-antigen includes lipopolysaccharides and a structurecontaining lipopolysaccharides from which Lipid A is excluded (e.g. astructure consisting of O-antigen or O-antigen and a corepolysaccharide). In case that a step of removing Lipid A is not used forthe purpose of efficient production, the vaccine composition of thepresent invention preferably comprises as an active ingredientlipopolysaccharides. Although there is no concern about side effects forthe vaccine composition of the present invention, the vaccinecomposition of the present invention preferably comprises as an activeingredient a structure containing lipopolysaccharides from which Lipid Ais excluded for higher safety.

(3) Monovalent Vaccine

The vaccine composition of the present invention includes a vaccinecomposition comprising as an active ingredient the aforementionedstructure derived from a single kind of Gram-negative bacteria(monovalent vaccine).

In case of a monovalent vaccine, Gram-negative bacteria may suitably beselected from the cells of Gram-negative bacteria as described above.Gram-negative bacteria are preferably Enterobacteriaceae or Pasteurella.Enterobacteriaceae is preferably Salmonella or E. coli. Salmonella ispreferably Salmonella belonging to O4, O7 and O9 Groups. Salmonellabelonging to O4 Group is preferably Salmonella Typhimurium, Salmonellabelonging to O7 Group is preferably Salmonella Infantis, and Salmonellabelonging to O9 Group is preferably Salmonella Enteritidis. E. coli ispreferably E. coli having O-antigen of O78. Pasteurella is preferablyHaemophilus, in particular, Haemophilus paragallinarum.

Thus, a monovalent vaccine of the present invention includes:

(A) a vaccine composition comprising as an active ingredient O-antigenderived from a single kind of Gram-negative bacteria;

(B) a vaccine composition comprising as an active ingredient O-antigenderived from a single kind of Enterobacteriaceae;

(C) a vaccine composition comprising as an active ingredient O-antigenderived from a single kind of Pasteurella;

(D) a vaccine composition comprising as an active ingredient O-antigenderived from a single kind of Salmonella;

(E) a vaccine composition comprising as an active ingredient O-antigenderived from a single kind of E. coli;

(F) a vaccine composition comprising as an active ingredient O-antigenderived from a single kind of Salmonella belonging to O4 Group;

(G) a vaccine composition comprising as an active ingredient O-antigenderived from a single kind of Salmonella belonging to O7 Group;

(H) a vaccine composition comprising as an active ingredient O-antigenderived from a single kind of Salmonella belonging to O9 Group;

(I) a vaccine composition comprising as an active ingredient O-antigenderived from Salmonella Typhimurium;

(J) a vaccine composition comprising as an active ingredient O-antigenderived from Salmonella Infantis;

(K) a vaccine composition comprising as an active ingredient O-antigenderived from Salmonella Enteritidis;

(L) a vaccine composition comprising as an active ingredient O-antigenderived from a single kind of E. coli having O-antigen of O78;

(M) a vaccine composition comprising as an active ingredient O-antigenderived from a single kind of Haemophilus; and

(N) a vaccine composition comprising as an active ingredient O-antigenderived from Haemophilus paragallinarum.

(4) Polyvalent Vaccine

The vaccine composition of the present invention includes a vaccinecomposition comprising as an active ingredient the aforementionedstructure derived from plural kinds of Gram-negative bacteria(polyvalent vaccine).

In case of a polyvalent vaccine, the kind of Gram-negative bacteria fromwhich a structure containing O-antigen is derived is preferably 2 ormore, 3 or more, 4 or more, or 5 or more. It is preferably 2 (bivalentvaccine), 3 (trivalent vaccine), 4 (4-valent vaccine), or 5 (5-valentvaccine).

A trivalent vaccine of Salmonella preferably comprises theaforementioned structures derived from Salmonella Typhimurium,Salmonella Infantis and Salmonella Enteritidis.

Gram-negative bacteria may suitably be selected from the cells ofGram-negative bacteria as described above. Gram-negative bacteria arepreferably Enterobacteriaceae or Pasteurella. Enterobacteriaceae ispreferably Salmonella or E. coli. Salmonella is preferably Salmonellabelonging to O4, O7 and O9 Groups. Salmonella belonging to O4 Group ispreferably Salmonella Typhimurium, Salmonella belonging to O7 Group ispreferably Salmonella Infantis, and Salmonella belonging to O9 Group ispreferably Salmonella Enteritidis. E. coli is preferably E. coli havingO-antigen of O78. Pasteurella is preferably Haemophilus, in particular,Haemophilus paragallinarum.

Thus, a polyvalent vaccine of the present invention includes:

(A) a vaccine composition comprising as an active ingredient O-antigensderived from two or more kinds of Gram-negative bacteria;

(B) a vaccine composition comprising as an active ingredient O-antigensderived from two or more kinds of Enterobacteriaceae;

(C) a vaccine composition comprising as an active ingredient O-antigensderived from two or more kinds of Pasteurella;

(D) a vaccine composition comprising as an active ingredient O-antigensderived from two or more kinds of Salmonella;

(E) a vaccine composition comprising as an active ingredient O-antigensderived from two or more kinds of E. coli;

(F) a vaccine composition comprising as an active ingredient O-antigensderived from Salmonella belonging to O4 Group and Salmonella belongingto O7 Group;

(G) a vaccine composition comprising as an active ingredient O-antigensderived from Salmonella belonging to O4 Group and Salmonella belongingto O9 Group;

(H) a vaccine composition comprising as an active ingredient O-antigensderived from Salmonella belonging to O7 Group and Salmonella belongingto O9 Group;

(I) a vaccine composition comprising as an active ingredient O-antigensderived from Salmonella belonging to O4 Group, Salmonella belonging toO7 Group and Salmonella belonging to O9 Group;

(J) a vaccine composition comprising as an active ingredient O-antigensderived from Salmonella Typhimurium and Salmonella Infantis;

(K) a vaccine composition comprising as an active ingredient O-antigensderived from Salmonella Typhimurium and Salmonella Enteritidis;

(L) a vaccine composition comprising as an active ingredient O-antigensderived from Salmonella Infantis and Salmonella Enteritidis;

(M) a vaccine composition comprising as an active ingredient O-antigensderived from Salmonella Typhimurium, Salmonella Infantis and SalmonellaEnteritidis;

(N) a vaccine composition comprising as an active ingredient O-antigensderived from two or more kinds of E. coli having O-antigen of O78; and

(O) a vaccine composition comprising as an active ingredient O-antigensderived from two or more kinds of Haemophilus.

(5) Mixed Vaccine

The vaccine composition of the present invention includes a vaccinecomposition comprising a monovalent vaccine or a polyvalent vaccinetogether with antigens different from the aforementioned structure(mixed vaccine).

Such antigens include an attenuated pathogen, an inactivated pathogen, aprotein, a peptide, a nucleic acid, and a virus-like particle. Themonovalent vaccine or the polyvalent vaccine of the present inventionmay be used as a mixed vaccine in combination with at least one vaccineselected from the group consisting of vaccines against other viruses(e.g. infectious bronchitis virus, infectious bursal disease virus,avian encephalomyelitis virus, Egg drop syndrome virus, Newcastledisease virus, Avian reovirus, avian influenza virus, Marek's diseasevirus, infectious laryngotracheitis virus, avian pneumovirus, andfowlpox virus), bacteria (e.g. Mycoplasma gallisepticum, Mycoplasmasynoviae, Clostridium septicum, Clostridium perfringens, Campylobacterjejuni, and Campylobacter fetus) and protozoa (e.g. Leucocytozooncaulleryi, Eimeria tenella, Eimeria maxima, and Eimeria necatrix).

(6) Concentration and Ratio of Active Ingredient

The vaccine composition of the present invention comprises as an activeingredient a structure containing O-antigen. A concentration of theaforementioned structure is determined using endotoxin unit (=EU) as anindex. EU is measured pursuant to 2-(6) step of measurement as describedbelow. A concentration of the aforementioned structure contained in avaccine composition, in case of a polyvalent vaccine, may vary dependentupon the kind of the aforementioned structure or may be the same. Aconcentration of the aforementioned structure is preferably at least5,400 EU/mL, and more preferably 54,000 EU/mL, for the respectivestructure.

In case of a polyvalent vaccine, a ratio of the aforementioned structure(EU ratio) may be either equal or different ratio. In case of atrivalent vaccine comprising as an active ingredient the aforementionedstructure derived from Salmonella Typhimurium, Salmonella Infantis, orSalmonella Enteritidis, a ratio of the aforementioned structure ispreferably Salmonella Typhimurium: Salmonella Infantis: almonellaEnteritidis (1:1:1).

(7) Subject for Administration

In accordance with the present invention, the shedding of cells into thececal droppings in chickens immunized with a trivalent vaccine wasreduced as in Example 1. Thus, the vaccine composition of the presentinvention may be used for birds. Birds include ones bred for commercialand non-commercial purpose. Examples of birds include Galliformes (e.g.chicken, quails, and turkey), Anseriformes (e.g. duck, and goose),Charadriiformes (e.g. gull, barred button quail, and plover),Columbiformes (e.g. pigeon), Struthioniformes (e.g. ostrich),Passeriformes (e.g. crow, finch, sparrow, starling, and swallow),Psittaciformes (e.g. parrot), Falconiformes (e.g. eagle, and falcon),Strigiformes (e.g. owl), Sphenisciformes (e.g. penguin), andPsittaciformes (e.g. parakeet, and parrot), preferably chicken.

(8) Route of Administration

The vaccine composition of the present invention has swelling at thesite of administration restrained to a level acceptable as a vaccine.Thus, a variety of routes of administration may be conceived for thevaccine composition of the present invention, including e.g. the leg,the breast, cervix, oral, rectal, percutaneous, enteric administration,intramuscular, subcutaneous, intramedullary injection, directintraventricular, intravenous, intraperitoneal, intranasal andintraocular injection. The leg is a convenient administration route of avaccine composition. Thus, a preferable route of administration is theleg.

(9) Carrier

The vaccine composition of the present invention may comprise apharmaceutically acceptable carrier. For a pharmaceutically acceptablecarrier, any carrier used for production of a vaccine may be usedwithout limitation. Specifically, a carrier includes saline, bufferedsaline, dextrose, water, glycerol, isotonic aqueous buffer and acombination thereof. In addition to this, the vaccine composition of thepresent invention may further comprise an emulsifying agent, apreserving agent (e.g. thimerosal), an isotonic agent, a pH adjuster, aninactivating agent (e.g. formalin), and an adjuvant. An adjuvant ispreferably oil adjuvant.

(10) Carrier Protein

A structure containing O-antigen may optionally be bound to a carrierprotein for the purpose of enhancing an immunization reaction whenadministered. Such a carrier protein includes diphtheria toxoid (DT),tetanus toxoid (TT), cholera toxin (CT) and CRM197. Binding to a carrierprotein may be done via a linker (e.g. SPDP or ADH) or the structure maydirectly be bound to a carrier protein. As an example, binding to acarrier protein (DT/TT/CT) via SPDP or ADH using cyanogen bromide isdisclosed (Non-patent reference 1).

2. Process for Preparing Vaccine Composition

The second embodiment of the present invention is a process forpreparing a vaccine composition for birds comprising as an activeingredient a structure containing O-antigen derived from Gram-negativebacteria.

The process of the present invention comprises a step of culturingGram-negative bacteria (step of culture); a step of releasing theaforementioned structure from cells contained in the solution after saidculture (step of release); a step of collecting the aforementionedstructure from the solution after said release (step of collection), anda step of preparing the solution after said collection to obtain avaccine composition (step of preparation). In addition to the abovesteps, the process may further comprise a step of inactivating cells(step of inactivation) and/or a step of emulsifying the aforementionedstructure (step of emulsification). Preferably, the step of inactivationis carried out after the step of culture and the step of emulsificationis carried out after the step of collection. Besides, the processpreferably comprises a step of measuring an amount of the aforementionedstructure (step of measurement) after the step of collection. In casethat the aforementioned structure is one that does not contain Lipid A,the process preferably comprises a step of removing Lipid A (step ofremoving Lipid A). The step of removing Lipid A is preferably carriedout after the step of collection.

Each of the respective steps is explained below. As occasion demands,the respective steps are not necessarily carried out in the orderindicated below, some steps may be omitted, or some steps may be carriedout repeatedly. For instance, the step of emulsification needs not becarried out when oil adjuvant is not used. The step of inactivation andthe step of release may be carried out simultaneously (e.g. sonicationand formalin treatment are done in both steps).

(1) Step of Culture

The step of culture may suitably be modified for the kind of culturemedium and growth condition (time, temperature, oxygen concentration,carbon dioxide concentration, pH, and salt concentration) depending onthe kind of Gram-negative bacteria. In case of Salmonella, the cells arecultured on TPB culture at 35° to 43° C. (preferably at 37° C.) for 8 to24 hours (preferably 16 hours). In case of E. coli, the cells arecultured on LB culture at 30° C. to 43° C. (preferably at 37° C.) for 8to 24 hours (preferably 16 hours).

(2) Step of Inactivation

The step of inactivation may suitably be modified depending on the kindof Gram-negative bacteria. For instance, it may be done with physicaltreatment (e.g. X ray radiation, heat treatment, or sonication) orchemical treatment (e.g. formalin treatment, mercury treatment, alcoholtreatment, or hydrogen treatment). Any of these treatments may be donealone or in combination thereof. Formalin treatment is preferable.

(3) Step of Release

The step of release may be any procedures providing antigenicity of theaforementioned structure is retained. The procedures and the conditionsmay suitably be selected depending upon Gram-negative bacteria or theproperty of the aforementioned structure. For instance, it includessonication, phenol treatment, mechanical treatment, freezing andthawing, compression and decompression, osmotic pressure, cell walldecomposition (e.g. lysozyme treatment disclosed in Non-patent reference1 and Non-patent reference 2), and surfactant treatment, either alone orin combination thereof, preferably cell wall decomposition, sonication,or phenol treatment.

Sonication may be conducted under any conditions providing antigenicityof the aforementioned structure is retained. For instance, it may bedone at 25° C. or less, preferably in ice water, for 5 to 30 minutes,preferably for 15 minutes. The solution after sonication may be subjectto centrifugation for removing impurities.

Phenol treatment may be conducted under any conditions providingantigenicity of the aforementioned structure is retained. For instance,it may be done with 45 to 100%, preferably 100%, phenol at 4 to 80° C.,preferably at 68° C. for 5 to 30 minutes, preferably for 15 minutes.During the procedure, a container may be stirred at regular timeintervals. The solution after the reaction may be subject tocentrifugation for removing impurities. The solution after phenoltreatment may be subject to dialysis with a suitable buffer (e.g. PBS).

Nucleolytic treatment (e.g. DNase treatment or RNase treatment) orproteolytic treatment (protease treatment) may be conducted after thestep of release, as disclosed in Non-patent reference 1 and Non-patentreference 2. Nucleic acids may be removed by ethanol fractionation.Removal of proteins and removal of fatty acid side chain of Lipid A maybe conducted by acetic acid treatment.

(4) Step of Collection

The step of collection may suitably be modified depending upon theproperty of the aforementioned structure. For instance,lipopolysaccharide is negatively charged as a whole as comprising a lotof phosphate groups and thus is easily adsorbed to a positively chargedsubstance. Therefore, the aforementioned structure may be collected byadsorption utilizing such property such as affinity chromatography andadsorption with a positively charged membrane. For instance, PolymyxinB, histidine, histamine, lysine, aminated poly(γ-methyl-L-glutamic acid)beads, and a positively charged membrane with introduced ammonium groupsmay be conceived for a positively charged substance.

Alternatively, the aforementioned structure may be collected byutilizing hydrophobicity of Lipid A. A hydrophobic substance includespolypropylene, polyethylene, polystyrene, and PTEE membrane. Forcollecting the aforementioned structure, it is also conceived that asubstance binding to lipopolysaccharide is utilized including ananti-LPS antibody, an anti-LPS factor from Limulus polyphemus, BPI(bactericidal permeability increasing) protein, CAP18 (cationicantibacterial protein of 18 kDa), and LBP (LPS-Binding-Protein).Chromatography with a carrier to which such a substance is bound may beused.

Collection of the aforementioned structure may be attained by usingcommercially available products. For instance, ET-clean (registeredtrademark; Chisso Corporation) may be used. Collection may also be doneby ultracentrifugation as disclosed in Non-patent reference 1.

(5) Step of Removing Lipid A

A structure containing O-antigen may be one that contains Lipid A (e.g.lipopolysaccharide) or one that does not contain Lipid A (e.g.O-antigen, O-antigen plus core polysaccharide). In case of a structurethat does not contain Lipid A, the step of removing Lipid A ispreferably incorporated.

The step of removing Lipid A includes, as an example, heat treatmentwith addition of an acid. An exemplary process includes treatment with1% acetic acid at 100° C. for 90 minutes (Non-patent reference 1). Thisprocess is combined with a process of isolating Lipid A as removed. Anexemplary process for isolating Lipid A includes ultracentrifugation(Non-patent reference 1).

(6) Step of Measurement

An exemplary process for measuring lipopolysaccharide may be carried asdescribed below. A solution of the aforementioned structures dilutedwith distilled water and reference standard of CSE-L set (E. coli O113:endotoxin derived from H10) are added to a microplate. LAL reagent ofEndospecy ES-50M Set (SEIKAGAKU BIOBUSINESS CORPORATION) is added. Theplate is covered for the reaction at 37° C. for 30 minutes. A mixture ofsodium nitrite/hydrochloric acid, an ammonium sulphamate solution, and amixture of N-(1-naphtyl)ethylenediaminedihydrochloride/N-methyl-2-pyrrolidone in Toxicolor System DIA Set(SEIKAGAKU BIOBUSINESS CORPORATION) are added and the microplate is wellshaken. Absorbance is measured at two wavelengths 545 nm and 630 nm(Molecular Devices Japan, VersaMax) to determine endotoxin.

(7) Step of Emulsification

The step of emulsification is a process of mixing together a solutioncontaining the aforementioned structure, oil and an emulsifying agent.An emulsifying agent includes Tween 80 (registered trademark), Tween 60(registered trademark), Brij 721 (registered trademark), Eumulgin B2(registered trademark), Arlacel 165 FL (registered trademark), Tefose1500 (registered trademark), Glucamate SSE20 (registered trademark),Surf hope C-1216 (registered trademark), Surf hope C-1811 (registeredtrademark), Surhope SE Pharma D-1816 (registered trademark), Surf hopeSE Pharma D-1616 (registered trademark), Span 60 (registered trademark),Olepal isostearique (registered trademark), Glucate SS (registeredtrademark), and Surf hope C-1205 (registered trademark), preferablysorbitan monooleate. Oil includes vegetable oil, mineral oil, animaloil, synthetic oil and silicone oil, preferably light liquid paraffin.

(8) Step of Preparation

The step of preparation is, in case of a polyvalent vaccine, a processof mixing compositions comprising the respective aforementionedstructures derived from the specific microbial cells as obtained in theabove steps. For instance, in case of a trivalent vaccine comprising asan active ingredient the aforementioned structures derived fromSalmonella Typhimurium, Salmonella Infantis and Salmonella Enteritidis,a composition comprising the aforementioned structure derived fromSalmonella Typhimurium, a composition comprising the aforementionedstructure derived from Salmonella Infantis and a composition comprisingthe aforementioned structure derived from Salmonella Enteritidis aremixed together.

The obtained vaccine may be used alone as a salmonella vaccine for birdsor used as a mixed vaccine in combination with at least one vaccineselected from the group consisting of vaccines against other viruses(e.g. infectious bronchitis virus, infectious bursal disease virus,avian encephalomyelitis virus, Egg drop syndrome virus, Newcastledisease virus, Avian reovirus, avian influenza virus, Marek's diseasevirus, infectious laryngotracheitis virus, avian pneumovirus, andfowlpox virus), bacteria (e.g. Mycoplasma gallisepticum, Mycoplasmasynoviae, Clostridium septicum, Clostridium perfringens, Campylobacterjejuni, and Campylobacter fetus) and protozoa (e.g. Leucocytozooncaulleryi, Eimeria tenella, Eimeria maxima, and Eimeria necatrix).

In case of a polyvalent vaccine and a mixed vaccine, a ratio of therespective compositions comprising the aforementioned structure (EUratio) may be either equal or a ratio of a specific composition may beincreased or decreased.

The present invention is explained in more detail by means of thefollowing Examples but is not limited thereto.

Example 1

(1) Preparation of Antigen

1) Step of Culture

On each 50 mL of LB medium (comprising sodium chloride 10 g, BactTryptone 10 g, Bact Yeast Extract 5 g in 1,000 mL of culture medium),Salmonella Enteritidis (hereinafter referred to as “SE”), SalmonellaInfantis (hereinafter referred to as “SI”), and Salmonella Typhimurium(hereinafter referred to as “ST”) were cultured (37° C., 16 to 24 hours,4×10⁸ to 4×10⁹ CFU/mL).

2) Step of Inactivation

Formalin was added to the respective culture at 0.4% formalin fortreatment at 37° C. for 16 hours to inactivate the cells.

3) Step of Release

The inactivated cells were subject to ultrasonic crushing or treatedwith phenol to release lipopolysaccharide.

Ultrasonic crushing (Branson, Sonifier 350) of the cells was carried outin ice water for 15 minutes. Then, centrifugation (TOMY SEIKO Co, Ltd.,10,000×g, 15 minutes) was performed and supernatant was used in the stepof collection.

The phenol treatment of the cells was carried out by mixing 20 mL of thecell solution after formalin inactivation with an equivalent volume of asaturated phenol solution and heating the mixture at 68° C. for 15minutes while stirring at every 3 minutes. Then, after the mixture wasleft to stand at 4° C. for 1 day, centrifugation (TOMY SEIKO Co, Ltd.,10,000×g, 15 minutes) was performed. For further removing impurities,supernatant was centrifuged (TOMY SEIKO Co, Ltd., 10,000×g, 15 minutes).Thereafter, supernatant was dialyzed with PBS for 3 days and used in thestep of collection.

4) Step of Collection

Each 30 mL of supernatant was added to ET-clean L column (ChissoCorporation, catalogue No. 20015). After washing of the column with aphosphate buffer containing 0.15M NaCl (NaCl 8.76 g in 1,000 mL ofsolution), lipopolysaccharide was eluted with a phosphate buffercontaining 2M NaCl (NaCl 117.54 g in 1,000 mL of solution).

(2) Determination of Endotoxin

A solution of the respective antigens diluted with distilled water andreference standard of CSE-L set (E. coli O113: endotoxin derived fromH10) were added to a microplate. LAL reagent of Endospecy ES-50M Set(SEIKAGAKU BIOBUSINESS CORPORATION) was added. The plate was covered forthe reaction at 37° C. for 30 minutes. A mixture of sodiumnitrite/hydrochloric acid, an ammonium sulphamate solution, and amixture of N-(1-naphtyl)ethylenediaminedihydrochloride/N-methyl-2-pyrrolidone in Toxicolor System DIA Set(SEIKAGAKU BIOBUSINESS CORPORATION) were added and the microplate waswell shaken. Absorbance was measured at two wavelengths 545 nm and 630nm (Molecular Devices Japan, VersaMax) to determine endotoxin unit.

(3) Step of Emulsification and Step of Preparation

Each 3.6 mL of the antigen solution was mixed and emulsified with 14.4mL of oil adjuvant (a mixture of light liquid paraffin, sorbitanmonooleate and polysorbate 80). An equivalent amount of the respectivevaccines were mixed together to prepare a salmonella vaccine comprisingthree kinds of lipopolysaccharides (trivalent LPS vaccine). For saidvaccine, a vaccine comprising each 54,000 EU/mL, a vaccine comprisingeach 5,400 EU/mL, and a vaccine comprising each 540 EU/mL, oflipopolysaccharides derived from SE, ST, or SI were prepared.

(4) Immunization Test

Each 0.5 mL of the respective vaccines was administered to 5-week oldSPF chickens at the lower thigh muscle. As a control, a trivalent mixedwhole-cells vaccine comprising inactivated whole cells of SE, ST, and SIwas used. The trivalent mixed whole-cells vaccine was prepared byculturing the respective cells, inactivating the cells, diluting thecells with PBS at 54,000 EU/mL of lipopolysaccharide, and mixing andemulsifying the inactivated cells with oil adjuvant. A group with noadministration of vaccine was also provided. The administration site wasobserved up till 10 weeks after administration and safety was assessed.Four weeks after immunization, the chicken were challenged by oral routwith Salmonella SE (9.1×10⁹ CFU/chicken), SI (2.3×10⁹ CFU/chicken), orST (1.2×10⁹ CFU/chicken) to investigate effectiveness. For assessingeffectiveness, feces were collected at 1, 4, 7, 10, and 14 days afterchallenge and the number of the shedding cells into cecal dropping wasmeasured by the method described below.

(5) Measurement of Number of Cells

The collected feces were diluted with HTT medium at 20% emulsion and 50μL of the emulsion was applied to DHL agar plate for culture (37° C., 16to 24 hours). The next day, the number of the emerged colonies wascounted to measure the number of cells in cecum feces (CFU/g). For thosesamples which gave no colony after this direct culture, enrichmentculture was performed for 1 day and the number of cells was determinedby the presence of colony (those samples which gave colony after thisculture had the number of cells of 50 CFU/g). For those samples whichgave no colony after enrichment culture for 1 day, delayed secondaryculture was performed and the number of cells was determined by thepresence of colony (those samples which gave colony after this culturehad the number of cells of 10 CFU/g and those samples which gave nocolony even after this culture had the number of cells of 0 CFU/g).

(6) Method of Assessment for Edema in Legs

The administration site was observed for 10 weeks and safety wasassessed with swelling score of legs. For swelling score, Score 1 waslight swelling (swelling at a portion of the lower thigh) at theadministration site, Score 2 was moderate swelling (swelling at a wholeof the lower thigh), and Score 3 was severe swelling (in addition toswelling at a whole of the lower thigh, dropsy is detected bypalpation). Score was decided by visual observation and palpation.

(7) Results

The results of observation of swelling in legs are shown in FIG. 1. Theresults obtained after challenge with ST are shown in FIG. 2. Theresults obtained after challenge with SE are shown in FIG. 3. Theresults obtained after challenge with SI are shown in FIG. 4. As shownin FIG. 1, alleviation in swelling in legs was proved for the trivalentLPS vaccine as compared to the trivalent mixed whole cell vaccine.Swelling in legs was further alleviated for the trivalent LPS vaccine inwhich impurities such as proteins were further removed by the step ofphenol extraction. With regard to effectiveness, the shedding of cellswas reduced significantly in all the challenge tests with ST, SE and SIfor the administration group of trivalent LPS vaccine as shown in FIG.3, FIG. 4 and FIG. 5 to affirm effective of the trivalent LPS vaccine.The shedding of cells was also reduced in the administration group oftrivalent LPS vaccine with phenol extraction to prove that a structurecontaining O-antigen (specifically, lipopolysaccharide) is useful as anantigen for a salmonella vaccine.

Example 2

(1) Preparation of Antigen

The vaccine prepared in Example 1 comprising each 54,000 EU/mL oflipopolysaccharides derived from SE, ST, or SI was mixed with OILVAX 7(registered trademark; The Chemo-Sero-Therapeutic Research Institute;comprising infectious bronchitis virus Nerima strain, TM strain,Mycoplasma gallisepticum, Egg drop syndrome virus, and infectious coryzaA and C (recombinant antigen)). The resultant mixture was 10-valentmixed LPS vaccine comprising each 7,200 EU/mL of lipopolysaccharidesderived from SE, ST, or SI, 10^(8.4) EID₅₀/mL or more of Newcastledisease virus, 10^(6.4) EID₅₀/mL or more of infectious bronchitis virusNerima strain, 10^(6.4) EID₅₀/mL or more of TM strain, 10^(7.4) EID₅₀/mLor more of Mycoplasma gallisepticum, 10^(6.7) EID₅₀/mL or more of Eggdrop syndrome virus, and 2.4 μg/mL or more of infectious coryza A and C(recombinant antigen).

(2) Immunization Test

Each 0.5 mL of the 10-valent mixed LPS vaccine prepared above wasadministered to 5-week old SPF chicken at the lower thigh muscle. As acontrol, the administration group of OILVAX 7 or the trivalent LPSvaccine (each 5,400 EU/mL of lipopolysaccharide from SE, ST, and SI) anda group with no administration of vaccine were provided. Swelling inlegs was observed up till 10 weeks after administration to assesssafety. Four weeks after immunization, the chicken were challenged byoral rout with Salmonella SE (9.1×10⁹ CFU/chicken), or SI (2.3×10⁹CFU/chicken) to investigate effectiveness. For assessing effectiveness,feces were collected at 1, 4, 7, 10, and 14 days after challenge and thenumber of the shedding cells into cecal dropping was measured by themethod of measuring the number of cells described in Example 1. Anextent of swelling was assessed as described in Example 1.

(3) Results

The results of observation of swelling in legs are shown in FIG. 1. Theresults obtained after challenge with SE are shown in FIG. 5. Theresults obtained after challenge with SI are shown in FIG. 6. As shownin FIG. 1, alleviation in swelling in legs was proved for the 10-valentmixed LPS vaccine when lipopolysaccharide was further purified by addingthe step of phenol extraction. As a result of observation of theinjection site up till 10 weeks after immunization, no swellingproblematic in the field causing dysstasia (Score 3) was seen for the10-valent mixed LPS vaccine comprising lipopolysaccharide not treatedwith phenol to affirm safety of the 10-valent mixed LPS vaccine. Withregard to effectiveness, the reduction in shedding of cells equivalentto the trivalent LPS vaccine was seen in the challenge tests with SE orSI to affirm effectiveness of the 10-valent mixed LPS vaccine.

INDUSTRIAL APPLICABILITY

A vaccine comprising as an active ingredient a structure containingO-antigen (e.g. lipopolysaccharide) derived from Gram-negative bacteriaallows for reduction in side effects at the injection site. Besides, bymixing the aforementioned structures derived from a plurality ofGram-negative bacteria, it becomes possible to manufacture a polyvalentvaccine and a mixed vaccine without increasing an amount of injection.

The invention claimed is:
 1. A composition comprising lipopolysaccharidefrom Salmonella, wherein the Salmonella is one or more Salmonella groupsselected from the group consisting of O4, O7 and O9, and wherein thecomposition comprises at least one purified lipopolysaccharide O-antigenfrom Salmonella Enteritidis, Salmonella Typhimurium, and SalmonellaInfantis, provided that said O-antigen is not comprised in a whole cell.2. The composition of claim 1, further comprising at least one of anantigen from Newcastle disease virus, an antigen from avian infectiousbronchitis virus, an antigen from Mycoplasma gallisepticum, an antigenfrom Egg drop syndrome virus, or an antigen from Haemophilusparagallinarum.
 3. The composition of claim 1, comprising the O-antigenin an amount of at least 5,400 EU/ml.
 4. The composition of claim 1,comprising the O-antigen in an amount of at least 54,000 EU/ml.
 5. Thecomposition of claim 1, comprising two of the purifiedlipopolysaccharide O-antigens.
 6. The composition of claim 1, comprisingthree of the purified lipopolysaccharide O-antigens.
 7. The compositionof claim 1, further comprising an adjuvant.
 8. The composition of claim1, wherein the O-antigen is not bound to a carrier protein.
 9. Thecomposition of claim 1, wherein the O-antigen is bound to a carrierprotein.
 10. The composition of claim 9, wherein the carrier protein isselected from the group consisting of diphtheria toxoid (DT), tetanustoxoid (TT), cholera toxin (CT) and CRM197.